In the medical field, the medical research field etc., certain components in blood (such as serum albumin, immunoglobulin, hepatitis virus, rheumatoid factor, and C-reactive protein) must be measured and various measurement methods have been developed and implemented for this purpose. In frequently used methods among these methods, immunological assay is becoming main stream and, in the immunological assay, blood collected and acquired from a patient or a subject (hereinafter referred to as whole blood) is centrifuged to acquire supernatant (serum or plasma), which is diluted with an appropriate buffer solution, and an antibody that specifically reacts with an analyte is used for detecting the analyte in the serum or plasma. Such assays include a method based on single radial immunodiffusion using a polyclonal antibody, as qualitative testing. Latex agglutination immunoassay and an immunoturbidimetric method are included as representative quantitative testing.
The needs of “wanting to perform various tests during examination of a patient” are recently increasing even in clinics and small hospitals and the tests are increasingly performed as point-of-care testing (POCT) instead of conventional subcontract testing. The representative examples of such POCT reagents include an immunochromatographic lateral .flow test strip. Since an operation of separating plasma and serum from blood is cumbersome and requires skill in the POCT field, testing using whole blood is desired. An assay of measuring an analyte in a whole blood sample by using immunochromatography is disclosed as, for example, a method as well as a reagent and a kit using a test strip fitted with a blood cell separation membrane (Patent Document 1). However, when plasma separated by this procedure is directly used for measurement by immunochromatography based on the principle of sandwich-type immune reaction, if the analyte is excessively present, it is problematic that a “hook phenomenon” (also referred to as a “prozone phenomenon”) occurs and causes apparent reduction in value although the highly-concentrated analyte is present in the sample. Therefore, whole blood is normally hemolyzed and diluted for measurement so as to remain within a predetermined measurement range. However, in this case, the concentration of the analyte is diluted by the blood cell volume and the measurement value is lowered as compared to the case of serum or plasma samples and, therefore, the measurement value must be corrected by using a hematocrit value (volume percent of red cell). The correction of the measurement value of an analyte using a hematocrit value (volume percent of red cell) will hereinafter also simply be referred to as hematocrit correction.
The problem of the apparent reduction in value is normally corrected by performing uniform multiplication by a correction coefficient acquired from an average hematocrit value of healthy individuals. However, the hematocrit value varies among individuals and the reference value thereof ranges from 39 to 52% in men and 35 to 48% in women. Therefore, an accurate analyte concentration cannot be acquired by the correction using multiplication by a uniform coefficient. Therefore, to perform accurate hematocrit correction, the correction must be performed with a hematocrit value separately measured by using the same sample used for measuring the analyte concentration.
The hematocrit value is conventionally obtained by a microhematocrit method based on a centrifuging method or by using an automated hemocytometer through calculations from the number of red blood cells and an average red blood cell volume. On the other hand, another method using conversion from a measurement result of whole blood into a measurement value in the case of measuring serum or plasma is reported as a method of converting blood measurement result and, in this method, a hemoglobin concentration (g/L) in whole blood is measured; a numerical value obtained by multiplying the acquired hemoglobin concentration by about 3/10 is adopted as a hematocrit value (%); and the hematocrit value is used for converting a measurement result from whole blood into a measurement value in the case of measuring serum or plasma (Patent Document 2). However, since the hemoglobin concentration and the hematocrit value must be obtained by a method different from an immunochromatographic assay, this method is cumbersome, requires time and cost, and therefore cannot satisfy needs of testing in the POCT field.
Therefore, a method of measuring an analyte and hemoglobin at the same time based on immunochromatography is desired; however, since the hemoglobin concentration in whole blood is normally several dozen g/L to 200 g/L, which is very high concentration, 10,000- to 100,000-times dilution is required for an assay based on the principle of a normal sandwich-type immunoassay. In this method, a large amount of a diluting solution is required for performing the dilution at one step, leading to deterioration of measurement accuracy. A method using multistep dilution problematically lacks practicality for a testing method in the POCT field. To solve these problems, desired is an immunochromatographic assay that is capable of measurement, with a hemolytic dilution operation of whole blood by a factor of at most 50 to 400.